We will develop a sequential labeling and imaging strategy for single-molecule localization based super-resolution microscopy allowing the best currently available fluorophore, Alexa647, to be used to image an arbitrary number of cellular targets, providing a robust 'multi-color' super-resolution strategy. Imaging will proceed sequentially, using Alexa647 labelled primary antibodies. After each imaging cycle, the dye will be removed, and the next target in the sample will be labelled and then imaged. We will investigate three methods for sequential super-resolution (SR) imaging. Sequential SR Imaging through Photo-bleaching: Immunofluorescence labeling will be performed with Alexa647 labeled primary antibodies. After each super-resolution imaging cycle, the imaging buffer will be replaced by PBS and the cell irradiated with high-intensity light for approximately 5 to 10 minutes. This will photo-bleach any existing Alexa647 dyes from the last target. The next target will then be labelled using standard Immunofluorescence protocols, imaged, then bleached. Sequential SR Imaging using Restriction Enzymes: Primary antibodies will be pre-conjugated with short sequences of dsDNA or hairpins. The sequence will be designed such that it is easily cleaved by a restriction enzyme. The fluorophore will be attached at the end of the oligo such that digestion allows the fluorophore to separate from the antibody and be washed away. Sequential SR Imaging using Sequential Hybridization and Restriction: Primary antibodies will be pre-conjugated with short ssDNA oligonucleotides. The antibody for each target will have an oligo with a different sequence. Before any imaging, all cellular targets will be labelled with the corresponding antibodies. Sequential imaging will begin with a hybridization step where the complementary strand to one of the oligonucleotides is introduced into the sample. After hybridization and a washing step, SR imaging will be performed. After imaging, a restriction enzyme will be used to cleave the dsDNA, and the fluorophore will be washed away, similar to the above procedure.